The aim of our project remains to understand effects of glucocorticoid hormones on gene expression. We are investigating two responses: the lysis of T-lymphoma cells and the induction of fibronectin in a fibrosarcoma cell line. Our studies of glucocorticoid-induced lymphocytolysis have been done so far with murine lymphoid cell lines. In recent months we have extended our work to the human acute lymphoblastic leukemic cell line CEM. We have examined the basis for glucocorticoid resistance in a subclone of that line, CEM-C1, known to contain functional glucocorticoid receptor. Treatment of CEM-C1 cells with the DNA-demethylating agent 5-aza-2' deoxycytidine yielded glucocorticoid-sensitive subclones at high frequency (approximately 10%). This indicates that DNA methylation is responsible for inactivating the expression of a "lytic" function required for glucocorticoid-sensitivity in these cells. We have followed up on our observation that glucocorticoids increase 10-\to 20-fold the rate of fibronectin biosynthesis in HT1080 fibrosarcoma cells. Using a human fibronectin cDNA clone to probe Northern blots, we found that this probe hybridizes with a high molecular weight mRNA from dexamethasone-treated HT1080. This mRNA is present at a much lower level in untreated HT1080 cells and is undetectable in CEM cells, which do not produce fibronectin. By "slot" blot analysis of total cellular RNA, we found a 16-\to 32-fold increase in fibronectin RNA level after hormone treatment. Moreover, this induction is inhibited by a glucocorticoid antagonist, RU486. This study demonstrates that the dexamethasone-induced stimulation of fibronectin biosynthesis is the result of an increase in accumulated fibronectin mRNA mediated by the glucocorticoid receptor. Studies are currently in progress to examine whether this effect is due to an increased rate of transcription. (D)